Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, there are few published reports of targeted gene inactivations in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. Since L. biflexa has a better transformation frequency than other species, we plan to optimize new techniques in this organism. In FY2015 we have begun to evaluate different systems that may affect the transformation effiencies of leptospires. The CRISPR/Cas system, present in L. interrogans but absent in L. biflexa, targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have demonstrated that the CRISPR/cas operon is transcribed during in vitro growth and have integrated part of the operon into L. biflexa and have shown that the genes are also transcribed in this heterologous host. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. Also, we have initiated a collaboration with Dr. Wiedenheft at Montana State University, who is an expert in the function and structure of CRISPR/Cas systems. We have obtained a number of Cas9-plasmid constructs that have been used to inactivate genes in a variety of organisms and we will tailor them to target specific Leptospira genes. The lamda red recombinase system has been used successfully in other bacteria to improve targeted mutagenesis. We are also assessing this system in L. biflexa, and if it appears promising, we will test it in the pathogen L. interrogans. We completed a proteomic map of in vitro cultivated L. biflexa in FY2015. This project identified highly abundant proteins from membrane- and soluble-fractions that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modifications including phosphorylation, methylation, and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID, and Daniel Sturdevant and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.